The present invention relates generally to the manipulation of genetic materials and more particularly to the manufacture of specific DNA sequences useful in recombinant procedures to secure the production of improved variants of a polypeptide identified as natural thaumatin I.
Thaumatin is an extremely sweet-tasting protein produced in the arils of the fruit of the African shrub Thaumatococcus daniellii Benth. The fruit traditionally has been used in West Africa as a sweetener of palm wine, corn bread, and sour fruit. Thaumatin, which is about 5,000 times sweeter than sucrose on a weight basis, is produced in at least five forms: thaumatins I, II, a, b and c. These proteins, named in the order of elution from an ion exchange column [Higginbotham, et al., in Sensory Properties of Foods (Birch, et al., eds.), London: Applied Sciences, pp. 129-149 (1977)], have molecular weights of approximately 22 kilodaltons. Thaumatins I and II are nearly identical proteins, each consisting of a single unmodified polypeptide chain, 207 amino acid residues in length.
Thaumatin is a non-toxic, low-calorie and noncariogenic protein which elicits profound sweet taste responses. These properties suggest a stable interaction between the proteins and human taste receptors. Therefore, thaumatin has potential for use as a sugar substitute, food additive, a sweetness receptor probe and a tool for further elucidation of the taste response.
A plentiful supply of pure thaumatin is required to utilize the protein as a possible food additive and research tool. Because the thaumatin plant requires a tropical climate and insect pollination for successful fruit propagation, there are considerable difficulties involved in greenhouse cultivation of the fruit.
Iyengar disclosed an amino acid sequence for thaumatin I which is shown in Table 1 below [Iyengar, et al., Eur. J. Biochem., 96, 193-204 (1979)].
TABLE 1 __________________________________________________________________________ ##STR1## ##STR2## ##STR3## ##STR4## ##STR5## ##STR6## ##STR7## ##STR8## ##STR9## ##STR10## ##STR11## ##STR12## ##STR13## ##STR14## ##STR15## __________________________________________________________________________
The amino acid sequence for thaumatin II has been deduced from its nucleotide sequence [Edens, et al., Gene, 18, 1-12 (1982)] and a gene for thaumatin II has been cloned from messenger RNA-derived cDNA. The five amino acids in the thaumatin II sequence which differ from the thaumatin I sequence above are the following: lysine instead of asparagine at residue 46; arginine instead of serine at residue 63; arginine instead of lysine at residue 67; glutamine instead of arginine at residue 76; and aspartic acid instead of asparagine at residue 113. Sequence analysis also indicated that thaumatin II is initally translated as a precursor form, preprothaumatin, with both a 22 residue amino-terminal extension and an acidic, six-amino acid carboxy terminal tail. The amino terminal peptide was postulated as a secretion signal based on its hydrophobic character and a compartmentalization role was hypothesized for the carboxy terminal extension.
The Edens, et al. reference cited above notes that a polypeptide having the native sequence of preprothaumatin II has been microbially produced. More specifically, the reference and Verrips, et al., U.S. Pat. No. 4,771,000 disclose cDNA sequences coding for native mature thaumatin II and preprothaumatin II and also disclose cloning vehicles comprising the DNA sequences for use in transformation in microorganisms.
In co-owned and copending U.S. patent application Ser. No. 540,634 filed Oct. 11, 1983, the disclosure of which is herein incorporated by reference, techniques for the synthesis of manufactured genes coding for the amino acid sequence of thaumatin I as identified by Iyengar, et al. were disclosed, as were DNA microorganism transformation vectors, fusion genes, transformed microorganisms, and processes for expressing the manufactured gene and for securing the polypeptide product produced thereby. Specific manufactured genes of the application incorporated a number of codons "preferred" for expression in yeast host cells.
In co-owned and copending U.S. patent application Ser. No. 189,250, a continuation of Ser. No. 797,474 filed Nov. 13, 1985, the disclosure of which is hereby incorporated by reference, polypeptides and genes for their synthesis were disclosed having the amino acid sequence of [Asp.sup.113 ] thaumatin I and [Lys.sup.46, Asp.sup.113 ] thaumatin I, i.e., containing the continuous sequence of amino acid residues of natural thaumatin I as reported by Iyengar, et al. except for an aspartate amino acid residue substituted for asparagine in the 113th position from the amino terminal end of the polypeptide and optionally a lysine amino acid residue substituted for asparagine in the 46th position. Those polypeptides could be folded to a sweet conformation using an in vitro procedure. Recombinant produced thaumatin I having the published Iyengar, et al. amino acid sequence was not sweet and could not be folded to a sweet conformation.
While the recombinant produced [Asp.sup.113 ] and [Lys.sup.46, Asp.sup.113 ] thaumatin I polypeptides constitute improvements over the art, they share with plant derived thaumatin taste characteristics which may limit their use in some food products. Most significant of these characteristics is a lingering aftertaste unlike that exhibited by sugar (sucrose).
Of interest to the present invention is the disclosure of van der Wel, et al., Chemical Senses and Flavor, 2, 211-218 (1976). This reference discloses experiments in which lysine residues in thaumatin I were chemically modified by acetylation with acetic anhydride and by reductive methylation. At least four acetylated thaumatins were obtained with either one, two, three or four acetylated amino groups. In addition, a methylated thaumatin was produced having six dimethyl lysine residues and one monomethyl lysine residue. While the methylated thaumatin with seven modified lysine residues had a sweetness intensity practically equal to that of the original thaumatin, the sweetness intensity of the acetylated thaumatins decreased with the increasing number of acetylated amino groups. The sweet taste sensation disappeared completely when four acetylated lysine residues were introduced into the molecule. The reference suggested the possibility that a correlation between net charge and the sweetness intensity of the molecule may exist.
Of interest to another aspect of the present invention are references relating to polypeptide secretion signal sequences. Von Heijne, European J. Biochem., 133, 17-21 (1983), discloses a variety of eukaryotic signal sequences and notes that signal sequences typically contain two different, independent signals. The first signal is said to be in the form of a hydrophobic core while the second one is said to confer processing specificity. Oliver, Ann. Rev. Microbiol., 39, 615-48 (1985), relates to protein secretion in Escherichia coli and discloses a variety of bacterial secretion signals which are characterized as including three conserved features including (a) a positively charged amino terminus, (b) a hydrophobic core and (c) a highly conserved sequence adjacent to the processing site.
Kendall, et al., Nature, 321, 706-708 (1986), discloses the use of site-directed mutagenesis to produce a mutant signal sequence containing nine consecutive leucine residues in the hydrophobic core segment Sjostrom, et al., The EMBO Journal, 6, 823-831 (1987), relates to analysis of E. coli secretion signal sequences and discloses the presence of lysine residues in the amino terminal end of the sequence for a high number of E. coli signal sequences. Sjostrom, et al. note, however, that the signal peptides of eukaryotes rarely have sequence homologies.